CN101492731B - Quick PCR detection method for colloid bacillus cereus - Google Patents
Quick PCR detection method for colloid bacillus cereus Download PDFInfo
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- CN101492731B CN101492731B CN2008101625866A CN200810162586A CN101492731B CN 101492731 B CN101492731 B CN 101492731B CN 2008101625866 A CN2008101625866 A CN 2008101625866A CN 200810162586 A CN200810162586 A CN 200810162586A CN 101492731 B CN101492731 B CN 101492731B
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- pcr amplification
- thalline
- bacillus cereus
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Abstract
The invention discloses a quick PCR detection method of Bacillus mucilaginosus, including the following steps: 1) the pretreatment buffer solution of thalli is prepared, the prescription contains 10mmol/l Tris Cl and 1mmol/l EDTA, and the pH is 8.0; 2) samples are pretreated to obtain thalli; 3) the thalli is washed and suspended by the pretreatment buffer solution of thalli and has a warm bath for 3 to 10 minutes in boiling water, and DNA samples are obtained in supernatant after centrifugation for 10 to 15 minutes at a speed of 12000 to 15000 rpm at the temperature of 4 DEG C and are storedin an ice bath; 4) the DNA samples are used as templates, and specific PCR amplifies gyrB genetic fragments; 5) the PCR amplified product is detected and analyzed. In the invention, specific PCR detection method is used for quickly detecting and identifying Bacillus mucilaginosus.
Description
Technical field
The present invention relates to a kind of fast PCR detection method of colloid bacillus cereus.
Background technology
Colloid bacillus cereus (Bacillus mucilaginosus) claims silicate bacteria, this bacterium generally to have potassium decomposing, fixed nitrogen, phosphorus decomposing function again.China widely applies research since the fifties to this bacterium, is developed to microorganism potash fertilizer the nineties and uses at large-scale promotion of whole nation.At present, colloid bacillus cereus is produced bacterium as important microorganism potash fertilizer, is widely used in agriculture production, has effectively promoted the development of green agriculture.In recent years, colloid bacillus cereus also received much attention in the applied research of hot fields such as metallurgy, pottery, wastewater treatment, as was used for the ore desiliconization, effectively improved the ore quality; As additive, improve ceramic performance; As flocculation agent be used to purify liquid waste, removal or enriching heavy metal etc.The colloid bacillus cereus preparation is because of safety, environmental protection, easily realize advantages such as suitability for industrialized production having application potential.
At present a variety of again about the microbial inoculum of colloid bacillus cereus, manyly at present this bacterium is detected and identify with traditional isolation cultivation method.Isolation cultivation method is mainly according to the colony characteristics of colloid bacillus cereus, promptly transparent, protruding, hyaloid and viscosity etc., also other kind is as soil genus bacillus, Paenibacillus polymyxa etc. again for the bacterial classification that has similar morphological specificity according to the study, so obviously there are shortcomings such as detected result is unreliable, the time of lasting is long, process is loaded down with trivial details in traditional detection method.As seen, set up a kind of fast and convenient colloid bacillus cereus detection method to the detection of related products and study the ecological behavior of this bacterium in soil with significant.GyrB is the gene of coding gyrase B subunit, because this gene is singly to copy and have certain conservative property and degree of variation, more and more widely is applied to strain identification in recent years.The present invention designs Auele Specific Primer on the basis of the gyrB gene of measuring a plurality of bacterial strains of colloid bacillus cereus, set up the fast PCR detection method of colloid bacillus cereus.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of fast PCR detection method of colloid bacillus cereus is provided.
A kind of fast PCR detection method of colloid bacillus cereus comprises the steps:
1) preparation thalline pretreatment buffer liquid, its prescription is: 10mmol/l TrisCl, 1mmol/l EDTA, pH8.0;
2) sample pretreatment obtains thalline;
3) thalline is washed and suspension with the thalline pretreatment buffer liquid, behind the temperature bath 3-10min, through 4 ℃, the centrifugal 10-15min of 12000-15000rpm, supernatant is the DNA sample to the thalline suspension in boiling water, and ice bath is preserved;
4) be template with the DNA sample, specific PCR amplification gyrB gene fragment;
5) pcr amplification product detects and analyzes.
Described sample pretreatment obtains the thalline step: for the microbiological specimens of gemma form, gemma is suspended in the special culture solution of colloid bacillus cereus, 28-30 ℃ of temperature bathed 10-20min makes gemma sprout into thalline, and the centrifugal 3-5min of 5000-10000rpm collects thalline;
Described sample pretreatment obtains the thalline step: for the microbe colony sample, and direct picking colony; For liquid thalline sample, the centrifugal 3-5min of 5000-10000rpm collects thalline.
Described is template with the DNA sample, specific PCR amplification gyrB gene fragment step:
1) the specific PCR amplification reaction system is 20 μ l, comprising: 10 * damping fluid, 2 μ l, 0.3mmol.l
-1DNTP2 μ l, concentration is 0.1 μ mol.l
-1Upstream primer 1 μ l, concentration is 0.1 μ mol.l
-1Downstream primer 1 μ l, DNA sample 1-2 μ l, Taq enzyme 0.3-0.5U, deionized water 11.7-12.7 μ l;
2) the specific PCR amplification program is: 94-95 ℃ of pre-sex change 4-5min, and 94-95 ℃ of sex change 30-60s, 59-63 ℃ of renaturation 30-60s, 72 ℃ are extended 45-60s, after circulation 30-35 time, 72 ℃ of extension 8-10min.
Described upstream primer is F2:5 '-ACGGATATCTCCCAGACGTTCAT-3 '; Downstream primer is R5:5 '-ACGGGCACGCTGCGCCTGTACG-3 '.
Described pcr amplification product detects and analytical procedure:
1) get the pcr amplification product of 5-10 μ l, electrophoresis on the sepharose of 1.0-1.5%, behind the EB dyeing 10-15min, clear water rinsing 10-15min detects pcr amplification product on gel imaging system;
2), calculate the colloid bacillus cereus concentration of determining in the sample according to the having or not and pairing extension rate of pcr amplification product.
The fast PCR detection method of another kind of colloid bacillus cereus comprises the steps:
1) the high salt method of employing N,O-Diacetylmuramidase-SDS-is extracted the DNA in the pedotheque;
2) with DNA be template, landing-type pcr amplification gyrB gene fragment;
3) the PCR product detects with analytical procedure and is: the PCR product of getting 5-10 μ l, electrophoresis on the sepharose of 1.0-1.5%, behind the EB dyeing 10-15min, clear water rinsing 10-15min, on gel imaging system, detect pcr amplification product, determine in the sample whether colloid bacillus cereus is arranged according to having or not of pcr amplification product.
Described is template with the DNA sample, landing-type pcr amplification gyrB gene fragment step:
1) the PCR reaction system is 20 μ l, comprising: 10 * damping fluid, 2 μ l, 0.3mmol.l
-1DNTP 2 μ l, concentration is 0.1 μ mol.l
-1Upstream primer 1 μ l, concentration is 0.1 μ moll
-1Downstream primer 1 μ l, DNA sample 1-2 μ l, Taq enzyme 0.3-0.5U, deionized water 11.7-12.7 μ l;
2) landing-type pcr amplification program is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s, 69 ℃ of annealing 30s descend 1 ℃ at every turn, up to 60 ℃, and each temperature cycle 2 times, 72 ℃ are extended 30s; 95 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ of extension 30s circulate 18 times; 72 ℃ are extended 8min.
Described upstream primer is F2:5 '-ACGGATATCTCCCAGACGTTCAT-3 '; Downstream primer is R5:5 '-ACGGGCACGCTGCGCCTGTACG-3 '.
The present invention compared with prior art, detected result is accurate, the time is short, simple and efficient to handle, for detection, evaluation and the Dynamic Study on Growth thereof of colloid bacillus cereus provides effective ways.
Description of drawings
Fig. 1 is the electrophorogram of the gyrB gene specific pcr amplification product of liquid colloid bacillus cereus sample, the Marker of M:DNA; 10
-1-10
-5Expression dilution of sample multiple;
Fig. 2 is the electrophorogram of the gyrB gene specific pcr amplification product of soil microorganisms bacterium colony sample, the Marker of M:DNA; C
1-C
9Represent different bacterium colony samples;
Fig. 3 is the electrophorogram of the gyrB gene specific pcr amplification product of colloid bacillus cereus gemma sample, the Marker of M:DNA; S represents the gemma sample of colloid bacillus cereus;
Fig. 4 is the electrophorogram of the gyrB gene specific pcr amplification product of micro organism composite fertilizer sample, the Marker of M:DNA; 10
-1-10
-6Expression micro organism composite fertilizer dilution of sample multiple;
Fig. 5 is the electrophorogram of the gyrB gene specific pcr amplification product of topsoil gong sample, the Marker of M:DNA; S
1-S
3Represent different topsoil gong samples.
Embodiment
Embodiment 1
Present embodiment is the colloid bacillus cereus sample of tracer liquid, adopts following steps to carry out:
1) thalline obtains: the colloid bacillus cereus sample of liquid behind the centrifugal 5min of 10000rpm, with thalline pretreatment buffer liquid washing 2 times, is used thalline pretreatment buffer liquid suspension thalline again, and the mixing that fully vibrates obtains the thalline suspension; Adjust thalline suspension concentration to 10
6Cfuml
-1, and carry out serial dilution;
2) cellular lysate: get and respectively dilute bacterium liquid 100 μ l and place the 1.5ml centrifuge tube, temperature is bathed 5min in boiling water respectively, and through 4 ℃, the centrifugal 10min of 14000rpm, supernatant is the DNA sample, and ice bath is preserved;
3) be template with the DNA sample, specific PCR amplification gyrB gene fragment:
The a.PCR reaction system is 20 μ l: comprising: 10 * damping fluid, 2 μ l, 0.3mmoll
-1DNTP 2 μ l, concentration be 0.1 μ moll
-1Upstream primer 1 μ l, concentration be 0.1 μ moll
-1Downstream primer 1 μ l, Taq enzyme 0.3U, deionized water 11.7 μ l, DNA sample 2 μ l;
The b.PCR amplification program is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 60s, 60 ℃ of renaturation 45s, 72 ℃ are extended 45s, circulates after 30 times 72 ℃ of extension 10min;
C. upstream primer and downstream primer sequence are:
Upstream primer F2:5 '-ACGGATATCTCCCAGACGTTCAT-3 ';
Downstream primer R5:5 '-ACGGGCACGCTGCGCCTGTACG-3 '.
4) the PCR product detects and analyzes: get the PCR product of 10 μ l, and electrophoresis on 1.0% sepharose, behind the EB dyeing 10min, clear water rinsing 10min detects pcr amplification product on gel imaging system.The result is at colloid bacillus cereus 10
1-10
3Diluent bacterium liquid in all detect the specific PCR amplified production (seeing accompanying drawing 1) of about 500bp, calculating thalline suspension relative concentration is 10
6Cfuml
-1
The test sample of present embodiment is an isolated microbe colony from soil, adopts following steps to carry out:
1) thalline obtains: a small amount of microbial bacteria of picking falls within the 1.5ml centrifuge tube, with thalline pretreatment buffer liquid washing 1 time, use thalline pretreatment buffer liquid suspension thalline again after, the mixing that fully vibrates obtains the thalline suspension;
2) cellular lysate: get above thalline suspension 100 μ l and place the 1.5ml centrifuge tube, with each bacterium liquid respectively in boiling water temperature bathe 3min, through 4 ℃, the centrifugal 15min of 12000rpm, supernatant is the DNA sample, ice bath is preserved;
3) be template with the DNA sample, specific PCR amplification gyrB gene fragment:
The a.PCR reaction system is 20 μ l: comprising: 10 * damping fluid, 2 μ l, 0.3mmoll
-1DNTP 2 μ l, concentration be 0.1 μ moll
-1Upstream primer 1 μ l, concentration are 0.1 μ moll
-1Downstream primer 1 μ l, Taq enzyme 0.5U, deionized water 12.7 μ l, DNA sample 1 μ l;
The b.PCR amplification program is: 95 ℃ of pre-sex change 4min, and 95 ℃ of sex change 30s, 61 ℃ of renaturation 60s, 72 ℃ are extended 60s, circulates after 30 times 72 ℃ of extension 10min;
C. upstream primer and downstream primer sequence are:
Upstream primer F2:5 '-ACGGATATCTCCCAGACGTTCAT-3 ';
Downstream primer R5:5 '-ACGGGCACGCTGCGCCTGTACG-3 '.
4) the PCR product detects and analyzes: get the PCR product of 10 μ l, and electrophoresis on 1.0% sepharose, behind the EB dyeing 10min, clear water rinsing 10min detects pcr amplification product on gel imaging system.The result has 3 samples to produce about 500bp specific PCR amplified production (seeing accompanying drawing 2) in 9 microbe colony samples that detected, and shows that these 3 samples are colloid bacillus cereus.
Embodiment 3
The test sample of present embodiment is the gemma of colloid bacillus cereus, adopts following steps to carry out:
1) gemma is sprouted: the gemma of getting colloid bacillus cereus is suspended in the colloid bacillus cereus special culture solution, and 30 ℃ of temperature are bathed 10min, make gemma sprout into thalline;
The prescription of colloid bacillus cereus special culture solution is: sucrose 0.5%, 0.1%K
2HPO
4, 0.02%MgSO
47H
2O, pH 7.5.
2) thalline preparation: the thalline after will sprouting, behind thalline pretreatment buffer liquid washing secondary, suspend with the thalline pretreatment buffer liquid, the mixing that fully vibrates obtains the thalline suspension;
3) cellular lysate: get above thalline suspension 200 μ l and place the 1.5ml centrifuge tube, temperature is bathed 8min in boiling water, and in 4 ℃, the centrifugal 10min of 15000rpm, supernatant is the DNA sample, and ice bath is preserved;
4) be template with the DNA sample, specific PCR amplification gyrB gene fragment:
The a.PCR reaction system is 20 μ l: comprising: 10 * damping fluid, 2 μ l, 0.3mmoll
-1DNTP 2 μ l, concentration be 0.1 μ moll
-1Upstream primer 1 μ l, concentration are 0.1 μ moll
-1Downstream primer 1 μ l, Taq enzyme 0.3U, deionized water 12.2 μ l, DNA sample 1.5 μ l;
The b.PCR amplification program is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 45s, 62 ℃ of renaturation 45s, 72 ℃ are extended 50s, circulates after 30 times 72 ℃ of extension 10min;
C. upstream primer and downstream primer sequence are:
Upstream primer F2:5 '-ACGGATATCTCCCAGACGTTCAT-3 ';
Downstream primer R5:5 '-ACGGGCACGCTGCGCCTGTACG-3 '.
5) the PCR product detects and analyzes: get the PCR product of 10 μ l, and electrophoresis on 1.0% sepharose, behind the EB dyeing 10min, clear water rinsing 10min detects pcr amplification product on gel imaging system.The result detects the specific PCR amplified production (seeing accompanying drawing 3) of about 500bp in the gemma sample of colloid bacillus cereus.
Embodiment 4
The test sample of present embodiment is the micro organism composite fertilizer that contains colloid bacillus cereus, adopts following steps to carry out:
1) gemma is sprouted: get microbiobacterial agent 5ml, through the centrifugal 3min of 5000rpm, gemma is suspended in the colloid bacillus cereus special culture solution, 28 ℃ of temperature are bathed 20min, make gemma sprout into thalline;
2) thalline preparation: the thalline after will sprouting, behind thalline pretreatment buffer liquid washing secondary, suspend with the thalline pretreatment buffer liquid, the mixing that fully vibrates obtains the thalline suspension, and carries out serial dilution;
3) cellular lysate: the thalline diluent 200 μ l that get after the above sprouting place the 1.5ml centrifuge tube, and temperature is bathed 10min in boiling water, and in 4 ℃, the centrifugal 12min of 14000rpm, supernatant is the DNA sample, and ice bath is preserved;
4) be template with the DNA sample, specific PCR amplification gyrB gene fragment:
The a.PCR reaction system is 20 μ l: comprising: 10 * damping fluid, 2 μ l, 0.3mmoll
-DNTP2 μ l, concentration be 0.1 μ moll
-1Upstream primer 1 μ l, concentration are 0.1 μ moll
-1Downstream primer 1 μ l, Taq enzyme 0.3U, deionized water 11.7 μ l, DNA sample 2 μ l;
The b.PCR amplification program is: 95 ℃ of pre-sex change 5min, and 95 ℃ of sex change 50s, 62 ℃ of renaturation 45s, 72 ℃ are extended 60s, circulates after 30 times 72 ℃ of extension 10min;
C. upstream primer and downstream primer sequence are:
Upstream primer F2:5 '-ACGGATATCTCCCAGACGTTCAT-3 ';
Downstream primer R5:5 '-ACGGGCACGCTGCGCCTGTACG-3 '.
5) the PCR product detects and analyzes: get the PCR product of 10 μ l, and electrophoresis on 1.0% sepharose, behind the EB dyeing 12min, clear water rinsing 12min detects pcr amplification product on gel imaging system.The result is 10
1-10
5Dilution bacterium liquid in all detect the specific PCR amplified production (accompanying drawing 4) of about 500bp, the relative concentration that calculates colloid bacillus cereus in the micro organism composite fertilizer sample is 10
8Cfuml
-1
Embodiment 5
The test sample of present embodiment is a topsoil gong, adopts following steps to carry out:
1) pedotheque pre-treatment: take by weighing the 300mg pedotheque, add 1.8ml damping fluid vortex vibration 10min, 4 ℃, the centrifugal 5min of 12000rpm remove supernatant;
Buffer formulation: 50mM Tris, 20mM EDTA, 100mM NaCl, 0.01g/ml PVPP, pH10.
2) DNA extraction: in pretreated pedotheque, add 0.05g quartz sand and 300 μ l DNA extraction damping fluid vortexs vibration 10min, add 10 μ l100mgml
-1N,O-Diacetylmuramidase, mixing, 37 ℃ of temperature are bathed 30min; Add 300 μ l SDS damping fluids, mixing, 65 ℃ of temperature are bathed 30min, 4 ℃, the centrifugal 15min of 8000rpm, liquid phase layer in the middle of collecting adds isopyknic chloroform/primary isoamyl alcohol (24:1), gentle mixing 3min; 4 ℃, the centrifugal 15min of 15000rpm collect supernatant, add the 3M NaAC (pH 5.2) of 0.1 times of volume, behind the gentle mixing, add the Virahol of 0.6 times of volume ,-20 ℃ of precipitation 1h; 4 ℃, the centrifugal 15min of 15000rpm abandon supernatant; With 70% washing with alcohol once, behind the vacuum-drying 3min, add 30 μ l TE dissolving and be the DNA sample.
The DNA extraction buffer formulation: 100mM Tris, 100mM EDTA, 200mM NaCl, 1%PVP, 2%CTAB, pH 8.0;
The SDS buffer formulation: 100mM Tris, 200mM NaCl, 3%SDS, pH 9.0.
3) be template with the DNA sample, landing-type pcr amplification gyrB gene fragment step:
The a.PCR reaction system is 20 μ l: comprising: 10 * damping fluid, 2 μ l, 0.3mmoll
-1DNTP2 μ l, concentration be 0.1 μ moll
-1Upstream primer 1 μ l, concentration are 0.1 μ moll
-1Downstream primer 1 μ l, Taq enzyme 0.3U, deionized water 11.7 μ l, DNA sample 2 μ l;
B. landing-type pcr amplification program is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s, 69 ℃ of annealing 30s descend 1 ℃ at every turn, up to 60 ℃, and each temperature cycle 2 times, 72 ℃ are extended 30s; 95 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ of extension 30s circulate 18 times; 72 ℃ are extended 8min.
C. upstream primer and downstream primer sequence are:
Upstream primer F2:5 '-ACGGATATCTCCCAGACGTTCAT-3 ';
Downstream primer R5:5 '-ACGGGCACGCTGCGCCTGTACG-3 '.
4) the PCR product detects and analyzes: get the PCR product of 10 μ l, and electrophoresis on 1.0% sepharose, behind the EB dyeing 15min, clear water rinsing 15min detects pcr amplification product on gel imaging system.The result all detects the specific PCR amplified production (seeing accompanying drawing 5) of about 500bp in 3 pedotheques.
Claims (5)
1. the fast PCR detection method of a colloid bacillus cereus is characterized in that comprising the steps:
1) preparation thalline pretreatment buffer liquid, its prescription is: 10mmol/l TrisCl, 1mmol/l EDTA, pH 8.0;
2) sample pretreatment obtains thalline;
3) thalline is washed and suspension with the thalline pretreatment buffer liquid, behind the temperature bath 3-10min, through 4 ℃, the centrifugal 10-15min of 12000-15000rpm, supernatant is the DNA sample to the thalline suspension in boiling water, and ice bath is preserved;
4) be template with the DNA sample, specific PCR amplification gyrB gene fragment;
5) pcr amplification product detects and analyzes;
Described is template with the DNA sample, specific PCR amplification gyrB gene fragment step:
1) the specific PCR amplification reaction system is 20 μ l, comprising: 10 * damping fluid, 2 μ l, 0.3mmoll
-1DNTP 2 μ l, concentration is 0.1 μ moll
-1Upstream primer 1 μ l, concentration is 0.1 μ moll
-1Downstream primer 1 μ l, DNA sample 1-2 μ l, Taq enzyme 0.3-0.5U, deionized water 11.7-12.7 μ l;
2) the specific PCR amplification program is: 94-95 ℃ of pre-sex change 4-5min, and 94-95 ℃ of sex change 30-60s, 59-63 ℃ of renaturation 30-60s, 72 ℃ are extended 45-60s, after circulation 30-35 time, 72 ℃ of extension 8-10min;
Described upstream primer is F2:5 '-ACGGATATCTCCCAGACGTTCAT-3 '; Downstream primer is R5:5 '-ACGGGCACGCTGCGCCTGTACG-3 '.
2. the fast PCR detection method of a kind of colloid bacillus cereus according to claim 1, it is characterized in that described sample pretreatment obtains the thalline step: for the microbiological specimens of gemma form, gemma is suspended in the special culture solution of colloid bacillus cereus, 28-30 ℃ of temperature bathed 10-20min makes gemma sprout into thalline, and the centrifugal 3-5min of 5000-10000rpm collects thalline.
3. the fast PCR detection method of a kind of colloid bacillus cereus according to claim 1 is characterized in that described sample pretreatment obtains the thalline step: for the microbe colony sample, and direct picking colony; For liquid thalline sample, the centrifugal 3-5min of 5000-10000rpm collects thalline.
4. the fast PCR detection method of a kind of colloid bacillus cereus according to claim 1 is characterized in that described pcr amplification product detects and analytical procedure:
1) get the pcr amplification product of 5-10 μ l, electrophoresis on the sepharose of 1.0-1.5%, behind the EB dyeing 10-15min, clear water rinsing 10-15min detects pcr amplification product on gel imaging system;
2), calculate the relative concentration of determining colloid bacillus cereus in the sample according to the having or not and pairing extension rate of pcr amplification product.
5. the fast PCR detection method of a colloid bacillus cereus is characterized in that comprising the steps:
1) the high salt method of employing N,O-Diacetylmuramidase-SDS-is extracted the DNA in the pedotheque;
2) be template with the DNA sample, landing-type pcr amplification gyrB gene fragment;
3) the PCR product detects with analytical procedure and is: the PCR product of getting 5-10 μ l, electrophoresis on the sepharose of 1.0-1.5%, behind the EB dyeing 10-15min, clear water rinsing 10-15min, on gel imaging system, detect pcr amplification product, determine in the sample whether colloid bacillus cereus is arranged according to having or not of pcr amplification product;
Described is template with the DNA sample, landing-type pcr amplification gyrB gene fragment step:
1) the PCR reaction system is 20 μ l, comprising: 10 * damping fluid, 2 μ l, 0.3mmoll
-1DNTP 2 μ l, concentration is 0.1 μ moll
-1Upstream primer 1 μ l, concentration is 0.1 μ moll
-1Downstream primer 1 μ l, DNA sample 1-2 μ l, Taq enzyme 0.3-0.5U, deionized water 11.7-12.7 μ l;
2) landing-type pcr amplification program is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 30s, 69 ℃ of annealing 30s descend 1 ℃ at every turn, up to 60 ℃, and each temperature cycle 2 times, 72 ℃ are extended 30s; 95 ℃ of sex change 30s, 60 ℃ of annealing 30s, 72 ℃ of extension 30s circulate 18 times; 72 ℃ are extended 8min;
Described upstream primer is F2:5 '-ACGGATATCTCCCAGACGTTCAT-3 '; Downstream primer is R5:5 '-ACGGGCACGCTGCGCCTGTACG-3 '.
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| CN101967456B (en) * | 2010-09-09 | 2012-07-04 | 中国农业科学院农业资源与农业区划研究所 | Bacillus mucilaginosus and culture method and culture medium thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1735694A (en) * | 2002-11-19 | 2006-02-15 | 莫比迪亚格公司 | Nucleic acid probes and broad-range primers from regions in topoisomerase genes, and methods in which they are used |
| CN101012263A (en) * | 2006-11-17 | 2007-08-08 | 中南大学 | General purpose primer for cloning bacteria gyrase-gyrB gene |
| CN101287843A (en) * | 2005-07-21 | 2008-10-15 | 森永乳业株式会社 | Method and kit for detection of microorganism |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1735694A (en) * | 2002-11-19 | 2006-02-15 | 莫比迪亚格公司 | Nucleic acid probes and broad-range primers from regions in topoisomerase genes, and methods in which they are used |
| CN101287843A (en) * | 2005-07-21 | 2008-10-15 | 森永乳业株式会社 | Method and kit for detection of microorganism |
| CN101012263A (en) * | 2006-11-17 | 2007-08-08 | 中南大学 | General purpose primer for cloning bacteria gyrase-gyrB gene |
Non-Patent Citations (3)
| Title |
|---|
| 侯晓丽等.分类及鉴别细菌的新靶标——gyrB基因.《国外医学 流行病学传染病学分册》.2005,第32卷(第1期),38-41. * |
| 曹凤明等.应用多重PCR鉴定微生物肥料常用芽孢杆菌.《微生物学报》.2008,第48卷(第5期),651-656. * |
| 李献梅等.促旋酶(gyrase) B亚单位基因gyrB在鉴别细菌近缘种中的应用.《微生物学报》.2008,第48卷(第5期),701-706. * |
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